This disclosure pertains to methods for isothermal strand displacement amplification that accomplishes efficient primer extension amplification with target specific primers and does not require pre-denaturation.
Isothermal amplification requires single stranded targets for efficient primer extension. Helicase dependent amplification of nucleic acids also requires helicase enzyme for unwinding double strands to allow amplification with a DNA polymerase (U.S. Pat. No. 7,282,328). Exponential strand displacement amplification (“SDA”) as described in U.S. Pat. No. 5,455,166 requires an initial denaturation of the target into single-stranded DNA (ssDNA), generation of hemiphosphorothioate sites which allow single strand nicking by restriction enzymes, and extension by a polymerase lacking 5′-3′ exonuclease activity. Raising the temperature of the reaction to approximately 95° C. to render double strands into single strands is required to permit binding of the primers to the target strands. State of the art SDA amplification requires the denaturation of the target at elevated temperature to yield ssDNA for strand displacement isothermal amplification.
The use of a nicking enzyme to cleave one of the strands of a target instead of the generation of hemiphosphorothioate sites in SDA amplification was described in (Ehses et al, J. Biochem. Biophys. Methods. 63:170-86 (2005)). The design of primers to reduce non-predictable byproducts was also described. Denaturation at 95° C. was required by Ehses et al. after the addition of target and before the addition of any enzymes. Nicking enzyme SDA amplification without denaturation of target at 95° C. was reported in U.S. Patent Application Publication No. 2009/0092967. However, a limitation of the latter method is that a limited number of nicking enzymes are available and quite often no natural nicking site is present in a target region of interest. An abasic site endonuclease amplification assay was disclosed in U.S. Patent Application Publication No. 2004/0101893. The use of this assay as a post amplification detection system in combination with other amplification systems was also disclosed. These assays require a denaturation step of dsDNA.
It is known in the art that double stranded (ds) nucleic acid can be denatured in different ways. Heat denaturation is state of the art to separate ds DNA into single strands. Native DNA denatures at about 85° C. (White, Handler and Smith, Principles of Biochemistry 5th Edition, McGraw-Hill Kogakush, Ltd, pages 192-197, 1993). Early on, it was established that primer extension in amplification required the binding of a primer to a single strand DNA strand. This was preferably achieved by heating the sample at about 95° C. (M Panaccio and A Lew. PCR based diagnosis in the presence of 8% (v/v) blood. Nucleic Acids Res., 19: 1151 (1991)). It was recently reported that Watson-Crick pairs in naked DNA spontaneously flip into Hoogstein pairs under ordinary conditions, suggesting that DNA breathes (Fran-Kamentskii. Artificial DNA; PNA & XNA, 2:1, 1-3 (2011)).
A few nucleases cut just one strand of DNA thereby introducing a nick into DNA (Besnier and Kong, EMBO Reports, 21: 782-786 (2001)). Most such proteins are involved in DNA repair and other DNA-related metabolism and cannot easily be used to manipulate DNA. They usually recognize long sequences and associate with other proteins to form active complexes that are difficult to manufacture (Higashitani et al., J. Mol. Biol., 237: 388-4000 (1994)). Single strand nicking endonucleases which nick only one strand of the DNA double strands and traditional restriction endonucleases are listed and updated in the REBASE Database (rebase.neb.com; Roberts et al., Nucl. Acids Res., 31: 418-420 (2003)). Engineering of a nicking endonuclease has been described (Xu et al, PNAS 98: 12990-12995 (2001)).
Other methods using isothermal amplification have been disclosed recently (Niemz et al., Trends in Biotechnol., 29:240-250 (2011)). However, these amplification methods also utilize thermal or other denaturation.